Bernard Khor, Ph.D. - US grants

DESCRIPTION (provided by applicant): CANDIDATE/TRAINING: My goal for this career development award is to obtain the necessary training to become an independent physician-scientist studying novel circuits that control regulatory T cell (Treg) differentiation and functio. I am a transfusion medicine fellow who earned a Ph.D. in Immunology studying the regulation of T cell receptor gene assembly. Subsequently, I began pursuing a specific interest in the differentiation of naive T cells into TH subsets. Building on foundational skills in molecular genetics, I now plan to add expertise and mentored research experience in cellular immunology, chemical biology and animal models of inflammation to further our understanding of Treg development and function in the context of inflammatory disease. My mentor is an immunologist leader in IBD genetics and biology and provides an unparalleled environment uniquely suited to the success of this project and my career. My research advisory committee brings all necessary additional expertise and my oversight committee comprises successful investigators who are excellent mentors. My prior research and clinical training, together with mentored research and coursework during the award period will build fundamental skills and highlight important findings in the regulation of Treg differentiation that I will follow up as an independent, R01-funded investigator with the long-term goal of understanding how T cell biology is modulated in the context of inflammatory disease. PROJECT: Tregs are critical anti-inflammatory cells, impaired development and/or function of which leads to inflammatory disease. Emerging data shows that Tregs typically exhibit organ-specific specializations to facilitate niche-relevant roles, calling nto question whether current approaches to expand Tregs ex vivo can adequately reproduce such functions. I hypothesize that local microenvironments contain the cues necessary to induce specializations required for niche-specific functions. Our pioneering unbiased chemical biology efforts identified novel small molecule enhancers of Treg differentiation, including harmine and 2 scaffolds from the Broad DOS collection. The experimental pipeline described in Specific Aim 1 highlights optimized enhancers based on these scaffolds. Harmine and the optimized DOS enhancers are used to address the core hypothesis in Specific Aim 2, that delivery of small molecule Treg enhancers to mucosal surfaces promotes Treg development and function locally and distally, both at steady state and using in vivo models of inflammation. In Specific Aim 3, we address novel and testable mechanistic hypotheses raised by pilot studies with harmine, including lineage plasticity, as well as the role of novel candidate genes (DYRK1a and DYRK2) and pathways (Creb and NF-kB1 signaling) in Treg differentiation. These studies have important impact on the conceptual approach to Treg-based therapy and are likely to highlight novel regulators of Treg differentiation, as well as point to multidisciplinary approaches to rapidly generate mechanistic hypotheses, enhancing our basic understanding of Treg biology in the context of inflammatory disease.

Project Summary/Abstract The goal of this project is to enhance our understanding of the IDG-eligible kinases DYRK1B, DYRK2, DYRK3 and DYRK4. Our preliminary data implicate at least one of these kinases in regulating differentiation of anti-inflammatory Tregs. Treg-modulating therapies are greatly needed, particularly in autoimmunity. We built a published pipeline to quantitate how genetic and chemical perturbations impact Treg differentiation, this showed that DYRK1A regulates Th17, but not Treg, differentiation. We now propose to use this pipeline to identify the Treg-regulating DYRK. This will illuminate a novel immune cellular phenotype for an IDG-eligible gene, develop validated tools to study IDG-DYRKs in primary cells, inform development of novel Treg-enhancing drugs and highlight patient subsets for precision therapy. Our long-term goal is to help develop better therapies, including inhibitors of specific DYRK family members, to treat autoimmunity. The overall objectives in this application are to (i) develop genetic tools to identify which DYRK family member regulates Treg differentiation, and (ii) interrogate functional and mechanistic features of DYRK-deficient Tregs. The central hypothesis is that an unidentified DYRK family member inhibits Treg differentiation. The rationale for this project is that identifying the Treg-regulating DYRK family member will offer a strong scientific framework to illuminate our understanding of understudied DYRK(s) as druggable regulators of autoimmunity pathobiology and establish a pipeline to illuminate other IDG-eligible genes. The central hypothesis will be tested in two specific aims: 1) Define how overexpression of IDG-DYRKs affects Treg differentiation, and 2) Define how IDG DYRK inhibitors and knockout of IDG-DYRKs affects Treg differentiation. The first aim will interrogate how overexpressing each DYRK family member in primary murine and human CD4+ T cells affects Treg differentiation. The second aim will mechanistically interrogate how knockout of each DYRK family member in primary murine and human CD4+ T cells affects Treg differentiation and function. These studies leverage our published experimental pipeline that quantitates how genetic or chemical perturbations impact T cell differentiation. Key innovative features of this proposal include studying the immune function of IDG-DYRKs and the use of primary immune cells. The proposed research is significant because it is expected to (i) reveal a new cellular phenotype for an understudied gene of interest to IDG, (ii) identify a novel druggable regulator of autoimmune pathobiology, thus directing future mechanistic and therapeutic studies, (iii) develop characterized tools to manipulate understudied DYRKs in other primary cells and biologic contexts and (iv) establish a scalable pipeline that can be used to rapidly interrogate all genes of interest to IDG for effects on differentiation into Tregs as well as other lineages including Th17, Th1 and Th2. Ultimately, this has the potential to broadly illuminate immune function of IDG genes and advance precision therapy of autoimmunity for people in the general population.